The present invention relates to methods and compositions useful for the induction of oral tolerance to a coadministered antigen in mammals.
Immunological antibody responses to pathogens are required to prevent infections in the body, whereas, immunological tolerance is a property of the immune system that allows for the discrimination of self from non-self. A breakdown in immunological tolerance to self antigens allows the onset of anti-self immunological responses through the generation of anti-self antibodies and/or cellular immune responses. This breakdown is responsible for auto-immune diseases seen in both humans and other mammals.
Allergic immune responses to allergens such as those classically observed in, for example, hay fever, reactions to insect bites and common food allergies is suppressed through the generation of immunological tolerance to the antigen responsible for the allergy, i.e., the allergen. Repeated exposure to a particular allergen through controlled administration of allergen can induce tolerance in some patients.
Oral tolerance has been characterized in the literature as a state of antigen-specific systemic immunological unresponsiveness or tolerance, which is induced by prior oral administration or feeding of antigen. (A. M. Mowat, Immunology Today, Vol. 8, No. 3, 1987, pp. 93-98.) Such a state of systemic hyporesponsiveness to an administered protein or antigen has been observed and reviewed in the art. (H. L. Weiner, Proc. Natl. Acad. Sci. USA, Vol. 91, 1994, pp. 10762-10765; and H. L. Weiner and L. F. Mayer, eds., Annals of NY Acad. Sci., Vol. 778, 1996, pp. xiii-xviii.)
It has been shown that oral co-administration of antigens with cholera toxin B subunit as a delivery agent provides an efficient transmucosal delivery system for induction of immunological tolerance. (J. B. Sun, et al., Proc. Natl. Acad. Sci. USA, Vol. 91, 1994, pp. 10795-10799; and C. Czerkinsky, et al., Annals NY Acad. Sci., Vol. 778, 1996, pp. 185-193.) In these studies sheep red blood cells (SRBC), horse red blood cells (HRBC) or purified human gamma-globulin (HGG) were used as antigen and covalently conjugated to the cholera toxin B (CTB) subunit delivery agent. The SRBC-CTB, HRBC-CTB or HGG-CTB were administered orally to mice to induce oral tolerance to these antigens.
One example of a inflammatory demyelinating autoimmune disease in humans is Multiple Sclerosis. Experimental Autoimmune (a.k.a. Allergic) Encephalomyelitis (EAE) is a paralytic disease of the central nervous system (CNS) that can be induced in animals by injection, together with Complete Freund""s Adjuvant, of brain or spinal cord homogenate, purified Myelin Basic Protein (MBP) or other purified encephalitogenic proteins (derived from brain or spinal cord) or synthetic peptides whose amino acid sequences resemble those of encephalitogenic components of CNS tissues. EAE is widely used as a model for human autoimmune inflammatory demyelinating disorders such as Multiple Sclerosis (J-B. Sun, et al, Proc. Nat""l Acad. Sci., USA, 93, 7196-7201 (1996)). Certain strains of animals display greater susceptibility to the disease, including Lewis rats and SJL/J mice. Cats, dogs, Guinea Pigs and rabbits may also be susceptible. The most common source of active encephalitogens is Guinea Pig brain or spinal cord.
The encephalitogen/adjuvant suspension is injected into the footpads of experimental animals, inducing the onset of disease symptoms within 10-12 days. Prevention or modulation of EAE symptoms has been achieved by induction of oral tolerance via oral administration of large, numerous doses of MBP either before or after induction of the disease. Generally, at least five oral doses are required. Determination of synergistic or immune enhancing agents to be administered together with MBP in order to reduce the number or magnitude of the MBP doses required to modulate the disease symptoms is desirable. If such agents could be identified, immunogenic tolerance to these and other types of autoimmune diseases could be promoted.
The present invention relates to methods and formulations for inducing oral tolerance in a mammal, comprising orally administering to the mammal a pharmaceutical formulation comprising an antigen and a delivery agent or agents comprising at least one derivatized amino acid or a salt thereof in an amount sufficient to induce oral tolerance. These delivery agents allow the administration of lower or fewer doses of antigen than are required to induce the same degree of systemic immune suppression with the antigen alone. The immune responses involved include, but are not limited to, systemic antibody production or delayed-type hypersensitivity reactions. In addition, the antigens for use in the induction of oral tolerance do not have to be covalently linked to the delivery agents.
It is believed that the foregoing delivery agents, when used in the proportions noted below, enhance the action of the antigens by increasing the proportion of ingested antigen which reaches the systemic circulation in its tolerogenic form. It may be that this is achieved by stabilization by the delivery agent of the tolerogenic form or fraction of the antigen in a configuration which may more easily cross the mucosal epithelium. It will be understood that the methods and compositions of the invention are not limited by the foregoing possible mode of action.
The invention relates to methods of inducing oral tolerance in a mammal wherein the deriviatized amino acid is comprised of an amino acid bearing a free carboxyl group, an amide linkage and a hydrophobic chain comprised of aromatic and/or aliphatic components.
A preferred embodiment of the invention relates to methods of inducing oral tolerance in a mammal wherein the deriviatized amino acid is an acylated amino acid compound of the formula 
Ar is a substituted or unsubstituted phenyl, 
R5 is C1 to C10 alkyl, C1 to C10 alkenyl, phenyl, naphthyl, (C1 to C10 alkyl)phenyl, (C1 to C10 alkenyl)phenyl, (C1 to C10 alkyl)naphthyl, (C1 to C10 alkenyl)naphthyl, phenyl (C1 to C10 alkyl), phenyl (C1 to C10 alkenyl), naphthyl (C1 to C10 alkyl) and naphthyl (C1 to C10 alkenyl);
R5 is optionally substituted with C1 to C4 alkyl, C1 to C4 alkenyl, C1 to C4 alkoxy, xe2x80x94OH, xe2x80x94SH and xe2x80x94CO2R6, cycloalkyl, cycloalkenyl, heteroalkyl, alkaryl, heteroaryl, heteroalkaryl, or any combination thereof; and
R6 is hydrogen, C1 to C4 alkyl or C1 to C4 alkenyl.
Another preferred embodiment of the invention relates to methods of inducing oral tolerance in a mammal wherein the deriviatized amino acid is a sulphonated amino acid compound of the formula
Arxe2x80x94SO2xe2x80x94(R4)xe2x80x94OHxe2x80x83xe2x80x83II
wherein Ar and R4 are as defined above.
Examples of the aforementioned derivatized amino acids are described in FIG. 1 below and include:
4-[4-(N-salicyloyl)]aminophenyl butyric acid (E352);
N-salicyloyl phenylalanine (E94);
4-[4-(N-benzenesulfonyl)]aminophenyl butyric acid (E198);
3-[4-(N-2,3-dimethoxybenzoyl)]aminophenyl propionic acid (E702);
10-(N-salicyloyl)amino decanoic acid (E597);
4-[4-(N-4 phenylbutyryl)]aminophenyl butyric acid (E445);
4-[4-(N-2 methoxybenzoyl)]aminophenyl butyric acid (E579);
3-[4-(N-2 methoxybenzoyl)]aminophenyl propionic acid (E594); and
4-[4-(N-phenoxyacetyl)]aminophenyl butyric acid (E623).
The present invention also relates to pharmaceutical formulations for inducing oral tolerance in a mammal, comprising an antigen and a delivery agent or agents comprising at least one derivatized amino acid or a salt thereof in an amount sufficient to induce oral tolerance. Preferably, the invention relates to pharmaceutical formulations for inducing oral tolerance, wherein the derivatized amino acid is administered at a dose of about 100-1000 mg per kg of the subject""s body weight, preferably at a dose of about 250-750 mg per kg of body weight.
Also contemplated are methods and pharmaceutical preparations incorporating an adjuvant or adjuvants with the antigen and delivery agent or agents. The formulations are particularly advantageous for inducing oral tolerance to antigens which otherwise would require large and/or chronic dosing of antigen to induce such tolerance and which, by themselves, do not pass or are not taken up in the gastrointestinal mucosa and/or are susceptible to chemical cleavage by acids and enzymes in the gastrointestinal tract. Such antigens include those associated with or responsible for the induction of auto-immune diseases, clinical (allergic) hypersensitivities, and allograft rejection, and subunits or extracts therefrom; or recombinantly generated whole proteins, subunits or fragments thereof; or any combination thereof.